Breast cancer development and progression involve the abnormality of multiple genes through genetic and epigenetic alterations. By conducting differential DNA sequencing and in situ hybridization, the aberrant expression of synuclein-gamma (SNCG) (Lavedan et al. Hum Genet. 1998 103:106-112), also referred to as Breast Cancer Specific Gene 1 (breast cancerSG1)(Ji et al. Cancer Res. 1997 57:759-764), has been linked to the disease progression of breast cancer.
SNCG along with synuclein-alpha (SNCA) and synuclein-beta (SNCB) belong to a family of intrinsically disordered proteins (Uversky, V. N. Protein Sci. 2002 11:739-756). Intrinsically disordered proteins are partially or completely unfolded under physiological or non-denaturing conditions. Although often traditionally described as “random coil,” many studies have shown disordered proteins to possess significant nonrandom structure, usually in the form of residual secondary structure (Elierzer et al. J. Mol. Biol. 2001 307:1061-1073; Yao et al. Biochemistry 2001 40:3561-3571) and hydrophobic clustering (Choy et al. J. Am. Chem. Soc. 2003 125:11988-11992). The recent recognition that intrinsically disordered proteins are more common than previously thought and can play important biological roles has challenged the traditional protein structure-function paradigm, which holds that polypeptide chains require compact, globular structure in order to function (Wright, P. E. and Dyson, H. J J. Mol. Biol. 1999 293:321-331; Tompa, P. Trends Biochem. Sci. 2002 27:527-533; Uversky et al. J. Mol. Recognit. 2005 18:434-384).
SNCG mRNA and protein are not expressed in normal breast tissue or tissues with benign breast diseases but are abundantly expressed in a high percentage of invasive and metastatic breast carcinomas (Ji et al. Cancer Res. 1997 57:759-764; Bruening et al. Cancer 2000 88:2154-2163; Wu et al. Cancer Epidemiol. Biomarkers Prev. 2003 12:920-925). A series of in vitro and in vivo functional studies have demonstrated that SNCG expression significantly stimulates proliferation (Liu et al. Breast Cancer Res. Treat. 2000 62:99-107; Lu et al. J. Biol. Chem. 2002 277:31364-31372; Jiang et al. Cancer Res. 2004 64:4539-4546; Jiang et al. Cancer Res. 2003 63:3899-3903), invasion and metastasis of breast cancer cells (Jia et al. Cancer Res. 1999 59:742-747).
SNCG has been shown to interact with BubR1, a mitotic checkpoint kinase required for the prevention of cell mitotic divisions following severe cell damage or mutation (Gupta et al. Oncogene 2003 22:7593-7599). The interaction of SNCG with BubR1 inhibits mitotic checkpoint control upon spindle damage and confers cellular resistance to anti-microtubule drugs such as nocodazole (Gupta et al. Oncogene 2003 22:7593-7599).
Nocodazole, a very commonly used inhibitor of microtubule assembly, experiences much reduced efficacy in SNCG-overexpressing MCF7 breast cancer cells, which is attributed to the SNCG-BubR1 interaction (Gupta et al. Oncogene 2003 22:7593-7599). The inhibitory effects of SNCG on checkpoint function are reduced when the cellular expression level of BubR1 is increased by ectopic over-expression (Inaba et al. Breast Cancer Res. Treat. 2005 94:25-35). Additional evidence shows that the interaction of BubR1 with CENP-E, which is critically required for mitotic checkpoint signaling, is impaired in the presence of SNCG, thereby suggesting that SNCG may inhibit BubR1 function by interfering with the BubR1 CENP-E interaction (Inaba et al. Breast Cancer Res. Treat. 2005 94:25-35). Taken together, these results suggest that BubR1 is a cellular target of natively unfolded SNCG, and the interaction of SNCG-BubR1 may represent a novel mechanism for inactivation of the mitotic checkpoint (Gupta et al. Oncogene 2003 22:7593-7599; Inaba et al. Breast Cancer Res. Treat. 2005 94:25-35).
Currently, microtubule inhibitors are used as first-line chemotherapeutic agents to treat patients with advanced or metastatic breast cancer (Valero et al. J. Clin. Oncol. 1998 16:3362-3368; Chan et al. J. Clin. Oncol. 1999 17:2341-2354). However, response rates to this class of drugs vary significantly. These microtubule-disrupting agents are believed to arrest cells in mitosis by triggering mitotic checkpoint activation, resulting in cells arrested in the mitotic phase without entering anaphase (Blajeski et al. J. Clin. Invest. 2002 110:91-99). Prolonged treatments with these agents lead to cell death by apoptosis (Shin et al. Cancer Cell 2003 4:483-497). Since the working mechanism of anti-microtubule drugs relies heavily on the normal function of the mitotic checkpoint machinery in which BubR1 is a critical component (Shin et al. Cancer Cell 2003 4:483-497), the inhibitory effect of SNCG on BubR1 function may be related to the induced resistance of breast cancer cells to microtubule inhibitors after exogenous expression of SNCG (Pan et al. J. Biol. Chem. 2002 277:35050-35060).
SNCG has also been disclosed to be a chaperone protein in the heat shock protein (Hsp)-based multiprotein complex for stimulation of ligand-dependent estrogen receptor (ER)-(signaling and stimulates hormone-responsive mammary and ovarian tumorigenesis (Jiang et al. Cancer Research 2004 64:4539-4546).
The ankyrin repeat, a 33 residue sequence motif, is one of the most frequently observed amino acid motifs in protein databases (Mosavi et al. Protein Science 2004 13:1435-1448). Twenty-four copies of this repeat are contained within the cytoskeletal protein ankyrin, hence its name. However, ankyrin repeats have been found in many proteins with a wide range of different functions.
SNCA, a protein with 55.9% sequence homology to SNCG, has been shown in a yeast two hybrid screen and in neurons to interact with synphilin-1, a protein containing several protein-protein interaction domains, such as ankyrin-like repeats and a coiled-coil domain (Engelender et al. Nat. Genet. 1999 22:110-114).